In this clinical trial grant proposal, we wish to expand upon observations made during a previous clinical trial in which IL-12 was shown to boost immune responses to peptide antigen- specific immunizations in patients with resected melanoma that were immunized with MART-1, gp100 and tyrosinase epitope peptides with incomplete Freund's adjuvant. Important questions that remain to be resolved include the optimal dose, schedule and adjuvant combination for peptide immunizations in patients with melanoma. In this proposal, in order to generate more potent and long-lived T-cell immunity against melanoma antigens, we will perform a three arm randomized phase II trial in HLA-A2.1 patients with resected stages III/IV melanoma at very high risk for relapse. Arm A will consist of eight subcutaneous vaccinations with a MART-1/gp100/tyrosinase peptide/IFA combination with IL-12 at 30 ng/kg over one year as the "standard" regimen. Patients in Arm B will receive IL-12 at 30 ng/kg mixed with alum and injected proximal to the peptides/IFA based on murine and human data showing that alum alters the pharmacokinetic profile and increases the adjuvant effect of IL- 12. Patients in Arm C will receive IL-12 at 30 ng/kg with alum and additional T-helper peptides based on human and murine data showing that the provision of both non-specific and specific T- cell help augments the generation of cytolytic T cells. The principal endpoints of these studies to be conducted at USC/Norris Cancer Center will be immune response to the antigen peptides and a quantitative and kinetic comparison of immune responses on the three arms of the trial. Skin testing with the peptides, ELISPOT and cytokine release by peptide stimulated CTL grown from peripheral blood, and precursor CTL frequency estimated from tetramer assays prior to, during and after finishing the vaccine regimen will be used to measure immune responses which will be correlated with time to relapse and survival as the clinical endpoints. A novel aspect of this proposal will be a direct functional, biochemical and phenotypic assessment of tumor antigen-specific T cells using peptide- tetramer column purification and sorting of T-cells from the peripheral blood of vaccinated patients, followed by measurement of function by lysis and cytokine release and phenotype by flow cytometry. Analysis of defects in signal transduction molecules on freshly isolated antigen-specific T cells from vaccinated patients will also be performed. The kinetics of development of T-cell immune reactivity will be measured by tetramer assays on peripheral blood before, at 6 and 12 months after initiating the vaccine regimen, and 6 and 12 months after the last vaccination. The results of those assays will be used to design randomized phase III trials for resected high risk melanoma and to develop strategies for overcoming antigen-specific unresponsiveness in melanoma patients.